Staphylococcus aureus readily forms biofilms in vivo, whether existing as a colonizer or when causing a spectrum of infections. S. aureus can also easily colonize and tenaciously grow as biofilms on a wide variety implants such as cardiac valves, catheters and prosthetic joints. Staphylococcal biofilms (SB) are refractory to antibacterial therapy, resistant to the host's immune system and hence, persist for long periods of time. Hence, understanding of the mechanisms by which SB evade the host's immune system and persist in vivo is critical. We put forth a novel hypothesis that staphylococcal superantigens (SSAg), a family of potent staphylococcal exotoxins, contributes to the immune evasion and persistence of SB by directly impairing the protective Th1/Th17 adaptive immune responses. SSAg are the most potent activators of the immune system. SSAg bind directly to MHC class II molecules on antigen presenting cells and activate 30-50% of CD4+ and CD8+ T cells irrespective of their antigen specificities. Given the biological properties of SSAg, slow growth of biofilms and the chronicity of SB infections, continued exposure to small amounts of SSAg could potentially cause chronic activation of a wide repertoire of T cells which can lead to their exhaustion, deviation and/or deletion through upregulation of negative costimulatory molecules such as PD-1, LAG-3, TIM-3 and CTLA-4 as shown in other chronic viral, parasitic infections or malignancies. In support of this hypothesis, our recent in vivo studies using wound-associated and catheter-associated biofilm infections have shown that SB do produce SSAgs. However, the kinetics of production of SSAg by SB in vivo and their extent of impact on the adaptive immune system is not fully known. Hence, in Specific Aim 1, we will ?Establish the temporal kinetics of production of SSAg by SB in vivo?. Subsequently, in Specific Aim 2, we will ?Explore the role of SSAg in immune evasion and persistence of SB in vivo?. These exploratory studies will be conducted using a panel of immunocompetent, immunodeficient and reporter mice transgenically expressing HLA-DR3 using the catheter-associated biofilm infection model. The reasons being, (i) Conventional mice are 1011 times more resistant to SSAg due to poor binding of SSAg to murine compared to human MHC (HLA) class II molecules. Therefore, our humanized mice transgenically expressing HLA-DR3 molecules respond robustly to SSAg. (ii) Catheters are one of the most widely implants and infection of catheters with S. aureus biofilms is very common. Overall, the teleological reasons as to why S. aureus would produce so many different SSAg with similar biological functions have been debated. Given that 80% of staphylococcal infections involve biofilms formation, either on artificial implants or living tissue, and a significant percentage of these isolates produce one or more SSAg, our hypothesis that SSAg may contribute to the growth and survival of staphylococcal biofilms is innovative and the knowledge gained from our study could have a profound impact on treatment/management of SBI and hence, highly significant.